(A) Experimental Laboratory Component: Using reverse genetics, low pathogenic avian influenza (LPAI) viruses (H5N1 and H6N1) with the minimal multibasic cleavage site (MBCS) motif will be generated and the intravenous pathogenicity index (IVPI) and 50% bird infectious dose (BID50) will be determined in chicken and compared to LPAI H5N1 and H6N1. Next, these viruses will be serially passaged (10x) in chicken to see whether the minimal MBCS will evolve into a MBCS and the highly pathogenic avian influenza (HPAI) phenotype. Deep sequencing (RML Research Technologies Section, Genomics Unit) will be used to analyze the cleavage site motif of viruses from chicken in each passage. This part of the project is still on hold due to the effects of the H5 moratorium and subsequent restrictions by NIAID, NIH. No work has been done on this project in fiscal year 2012/13 (Fouchier et al., Science 2012; Fouchier et al., Science 2012; Fouchier et al., Science 2013) . (B) Field Survey Component: Historically, the natural reservoir for all influenza A viruses were considered wild birds. However, the recent identification of a novel H17 influenza A virus in yellow-shouldered bats in Guatemala has put the focus on novel reservoirs besides wild birds. Certain influenza A viruses in particular are a direct threat to both animal and public health. In particular, Highly Pathogenic Avian influenza A viruses (HPAI H5 and H7 subtypes) and a wide variety of swine influenza viruses (H1, H2 and H3) are notorious for crossing the species barrier from animals to humans. Public health, animal health, agricultural productivity and food security in the Republic of Congo (RC) and the wider Congo basin region are directly threatened by outbreaks of influenza A viruses. In addition, the potential zoonotic transmission of influenza viruses to the human population could spark outbreaks and the emergence of a new pandemic. To date little or no information has been published on the prevalence, incidence and identity of animal influenza viruses, in particular avian influenza viruses, in RC and there are currently no surveillance programs for avian influenza viruses in place. Therefore, there is a real need to develop baseline information with reference to the circulation of avian influenza viruses. During the fiscal year 2012/13, the laboratory infrastructure has been established at the Laboratoire National de Sant Publique in Brazzaville (RC capital) and diagnostic/surveillance assays have been developed and implemented (Grolla et al., J Clin Virol 2012; Mombouli et al., Emerg Infec Dis 2013; Nisii et al., PLoS Path 2013). Surveillance studies have started in fiscal year 2012/13 and first results are currently analyzed and prepared for publication. As a site project, the set up was used to investigate the emergence of Chikunyunga virus in RC. The outbreak was associated with both Aedes aegypti and Aedes albopictus mosquitoes, whereas outbreaks in neighboring Gabon are predominantly associated with Aedes albopictus. Our study confirms the presence of Aedes albopictus in RC and its role as a potent vector in Chikungunya virus transmission in the region (Mombouli et al., Emerg Infec Dis 2013). This project will be continued by Dr. Vincent Munster, Viral Ecology Unit, LV, NIAID, NIH. Dr. Feldmann will no longer serve as the principal investigator for this project.